A scanning electron microscope study of oxolinic acid treatment of burn spot lesions of Macrobrachium rosenbergii

Aquaculture, 52 (1986) 157-171 Elsevier Science Publishers B.V., Amsterdam -Printed

A SCANNING ELECTRON MICROSCOPE STUDY OF OXOLINIC TREATMENT OF BURN SPOT LESIONS OF MACROBRACHIUM ROSENBERGII

A.A. EL-GAMAL’, D. MACINTOSH’

D.J. ALDERMAN’,

157

in The Netherlands

C.J. RODGERS*, JANE L. POLGLASE’”

ACID

and

‘Institute of Aquaculture, University of Stirling, Stirling, E’K9 4LA (Great Britain) ‘Ministry of Agriculture, Fisheries and Food, Directorate of Fisheries Research, Fish Diseases Laboratory, Weymouth, Dorset, DT4 SUB (Great Britain) *To whom correspondence

should be addressed.

(Accepted 6 December 1985)

ABSTRACT El-Gamal, A.A., Alderman, D.J., Rodgers, C.J., Polglase, J.L. and Macintosh, D., 1986. A scanning electron microscope study of oxolinic acid treatment of burn spot lesions of Macrobrachium rosenbergii. Aquaculture, 52: 157--171. Black burn spot exoskeletal lesions of bacterial aetiology are a frequent feature of crustaceans in intensive culture. The appearance under the scanning electron microscope of such lesions associated with Aeromonas hydrophila on Macrobrachium rosenbergii is described. The effect of oxolinic acid treatment on these lesions is discussed.

INTRODUCTION

The occurrence of exoskeletal lesions in marine and freshwater crustaceans, in natural populations and in artificial culture, is well known. The burnlike appearance of such lesions has naturally given rise to various common names, such as burn spot disease, black spot, rust disease, Brandenfleckenkrankheit and Schwarzfleckenkrankheit. These lesions have been shown to be due to a range of causes, principally nutrition, pollution or infection. Lightner et al. (1977) coined the term “Black Death” in penaeid shrimp for melanised haemocytic lesions which occur throughout the tissue of animals maintained on diets deficient in ascorbic acid. “Black Death” is also characterised by poor wound repair (Lightner et al., 1979). Doughtieet al. (1983) described burn spot lesions in grass shrimp, PaZaemonetes pugis, exposed to hexavalent chromium and Doughtie and Rao (1983) have investigated black gill lesions in shrimp exposed to a dithiocarbamate biocide. In a discussion of burn spot lesions on crabs from natural crustacean fisheries, Ayres and Edwards (1982) suggested that, although some authors have linked the incidence of crustacean exoskeletal lesions 0 Crown Copyright

158

with organic and industrial pollution (Schlotfeldt, 1972; Gopalan and Young, 1975; Young and Pierce, 1975; Roald et al., 1981), in natural stocks at least, the severity of shell necrosis is greater in areas where crabs live longer, i.e., in areas which are not fished intensively. Ayres and Edwards (1982) showed that polluted (e.g., sewage dumping) areas, which are not fished intensively, have higher incidences of exoskeletal lesions and that these areas have comparatively high numbers of older animals which moult infrequently. Crabs with a prolonged intermoult period have a greater chance of developing infectious burn spot lesions, thus resulting in the higher frequency of such lesions in polluted areas. Although many burn spot lesions have been shown to be of fungal aetiology (Burns et al., 1979), those of bacterial origin are the most common (Rosen, 1970; Sindermann, 1977; New and Singholka, 1982). Bacterial burn spot lesions tend to be erosive and penetrative and, where limbs or appendages are involved, autotomy may result. The appearance, and thus the value, of the harvested crustaceans will be reduced and, in severe cases, significant mortalities may occur. In the most recent review of diseases of Mucrobruchium culture, Brock (1983), in regard to bacterial shell disease, commented that “the use of chemicals is generally not recommended in the control of this disease . . . however, antibiotics have been reported to successfully control bacterial necrosis . . . in M. rosenbergii larvae in Tahiti”. In contrast, Lightner (1984), reviewing diseases of cultured penaeid shrimp in the same volume, found that several authors had reported the successful use of antibiotics such as oxytetracycline and furanace to control bacterial shell disease in penaeids. When severe burn spot lesions developed in an experimental population of M. rosenbergii held in the tropical prawn facility of the Institute of Aquaculture, Stirling, the opportunity was taken to examine the lesions under the scanning electron microscope and to investigate the effects of treatment with oxolinic acid. To determine whether there were any significant differences between the bacterial flora of the lesions on these “Institute of Aquaculture” prawns in heated Scottish water and the lesions on prawns from a non-laboratory tropical environment, a stock of adult prawns was obtained directly from Malaysia from the same site as that from which the Stirling prawns originally derived, Exoskeletal lesions on these animals were examined and bacteriologically sampled immediately on arrival in the U.K. MATERIALS

AND METHODS

Prawns The prawns used in the first part of this study were M. rosenbergii produced at a commercial prawn hatchery in peninsular Malaysia and imported into Britain in May 1982 as 60day-old hatched post-larvae already acclimated

159

to freshwater. These post-larvae were reared for 8 months in a 600-l capacity fibre-glass aquarium fitted with a water recirculation pump and a gravel and glass-fibre wool filter. Water conditions were maintained as follows: temperature 27-28’C, pH 7.5 and total hardness 80 mg/l. The water and filter media were changed as necessary to prevent the build up of waste products. The prawns were fed ad libitum with a diet of mussels (MyCiZus) with minced prawn and fish. After 6 months, an increasing number of animals developed exoskeletal burn spot lesions. In addition, adult M. rosenbergii were imported from peninsular Malaysia in March 1984 and, together with their transport water, were examined immediately on arrival in the U.K. bacterial flora: ~~~a~~n and identification Bacterial isolates were obtained from prawns by taking swabs directly from lesions. Each infected area was sampled before and after dipping the exoskeleton in absolute alcohol. The swabs were then used to inoculate tryptone soya agar, TSA, (Oxoid) plates, which were incubated at 22°C for 14 days, Any colonies developing were re-inoculated onto fresh TSA plates. This procedure was repeated until pure cultures were obtained. Presumptive identification of the purified bacterial strains was achieved using the primary diagnostic scheme of Cowan and Steel (1974). Identification was confirmed using API-20E strips, incubated at 37°C for 24 h. Sensitiuity testing Antibiotic sensitivity testing (except for oxolinic acid) was by standard Multodisks (Oxoid) applied to the surface of inoculated iso-sensitest agar, ISA, (Oxoid). The compounds tested included ampicillin, chloramphenicol, cloxacillin, colistin sulphate, co-trimoxazole, e~thromyc~, fu~zolidone, gentamycin, kanamycin, neomycin, nitrofurantoin, novobiocin, oxytetracycline, penicillin G, sulphafurazole and tetracycline. Sensitivity to oxolinic acid was determined by preparing 6-mm diameter discs of Whatman No. 3 filter paper impregnated with 10 pg/ml active compound (Austin et al., 1981). Plates for sensitivity testing were incubated at 22°C for 48 h and zones of ~hi~tion in the resultant bacterial lawns were taken as an indication of sensitivity, Bacterial growth reaching the edge of the. impregnated disc was recorded as resistance to the antibiotic concerned. Treatments Treatments were carried dut by. t~nsferring infected animals to a separate well-aerated tank containing 20 1 of 10 pg/ml oxolinic acid in water at 28°C. Animals undergoing experimental treatment were held in this tank for 1 h and were then transferred to a 60-l glass tank provided with an external

160

e____.‘ -c=.-

"x,-*. *

161

filter to enable continuous observation. Water conditions in the obse~ation tank were maintained in the same manner as, and as close as possible to, those in the rearing tanks, Microscopic investigations During a pre~min~ investigation, pieces of infected exoskeleton which had been fixed in 10% neutral buffered formalin were subsequently prepared for scanning electron microscopy. Since this fixation proved adequate, for consistency it was retained for the rest of the investigation. In the treatment study, infected areas were excised before and at regular intervals after treatment with oxolinic acid. Dehydration was achieved via two 30-min changes of Z,Z-d~ethoxyprop~e, 50% in acetone (v/v) at 20°C followed by 100% acetone (two changes, each of 30 mm). The specimens were then placed in a Polaron P1500 critical point dryer, infiltrated with liquid carbon dioxide for 1 h before being dried by passing through the critical point. Finally, the specimens were carefully orientated on scanning electron micrograph (SAM) carriers before being coated with gold p~la~um in a Polaron E5100 high resolution cooled sputter coater for 4 min at 1,500 V, 25 mA. Exammations were made using a JEM 1OOCX 4ASID electron microscope at accelerating voltages of 100 or 80 kV. RESULTS

Gi-ossappearance of lesions In the earliest stages, infection was evident in the form of black, necrotic lesions of the exoskeleton, primarily on the appendages. The initial lesions usually occurred on the antennal flagellum, the rostrum, the telson or on the ends of the pereiopods. Animals with this class of lesion were recorded as lightly or moderately infected. As the disease progressed, lesions slowly eroded the appendages and to a lesser extent the tergum edges resulting in extensive damage to the infected areas (Fig. 1). Animals showing a degree of infection which impeded normal behaviour were classed as severely infected. Necrotic lesions also appeared on the tergites in the form of black spots with depressed centres. Animals with such infections tended to die rapidly but those with the disease confined to the appendages were able to survive for several months. In the latter case, mortality was most likely to be due to prolonged interference with normal locomotory and feeding Fig. 1. Normal (lower) A& rosenbergii compared with severely burn spot infected animal (upper). Fig. 2. SEM of burn spot lesion of edge of swimmeret (X 300). Fig. 3. SEM of lesion on walking leg tip (X 100).

Pre-dip prawns1 Pre-dip prawns1 Pre-dip prawn S2 Post-dip prawn53 Post-dip prawns3 Postdip prawn 54

PRl

-

-

-

-

+

-

Gram stain

R

R

R

R

R

R

Shape

Primary tests

+

+

+

+

+

+

Motility

R, rod shaped; 0, oxidative; F, fermentative.

PR6

PR5

PR4

PR3

PR2

Source

Isolate code

F

F

F

F

o/--

0

O/F

+

+

+

+

+

+

Catalase

+

+

+

+

+

+

Oxidase

+

+

+

+

+

+

Growth at 37°C

7247126

7247127

7247125

7247127

1003004

2205004

API profile no.

Aeromonas

Aeromonas

Aeromonas

Aeromonas

Bacillus sp .

hydrophila

hydrophila

hydrophila

hydrophila

Pseudomonas fluorescens

Identification

Presumptive identification of bacterial isolates from prawn burn spot lesions (Institute of Aquaculture stock)

TABLE 1

163

behaviour. At the height of infection, exhibited varying degrees of infection.

nearly 85% of the stock animals

Light microscopy Small pieces of infected melanised exoskeleton were examined as fresh prep~ations under the light microscope. No evidence of any fungal infection was detectable. This suggested that a likely cause of the melanisation was bacterial infection. Bacterial isolates All plates inoculated with swabs taken from lesions prior to the alcohol dip were quickly overgrown by a number of bacterial isolates. In the first part of the study, following purification, the predominating strains from undipped exoskeletons were identified as Pseudomonas fluorescens, Aeromonas hydrophila and BaciUus sp. Plates inoculated from lesion material after the alcohol dip yielded only A. ~ydrophi~a {Table 1). In the second study with freshly imported prawns, the transport water was found to contain mainly A. hydrophiba (Table 2). The total viable count for the transport water was 1.63 X lo7 cells/ml after a transit time of 36 h. The prawns themselves were heavily but superficially covered by a mixed bacterial growth presumed to be a transport artifact, so that only alcoholdipped lesions are considered here. Plates inoculated from alcohol-dipped telson lesions predomin~tly yielded A. ~ydroph~~a and a single limb lesion gave Pseudomonas maltophilia (Table 2). The antibiotic sensitivities for the 7 strains of A. hydrophila (PR3, PR4, PR.5, PR6, PR7, PR8 and PRlO) are listed in Table 3. The strain of P. fluorescens (PRl) was sensitive to tetracycline at 10 and 50 #g/ml, gentamycin at 10 ggfml, colistin sulphate at 10 pgjml, kan~yc~ at 30 @g/ml, neomycin at 10 pg/ml and oxytetracycline at 10 &g/ml but resistant to all of the other antibiotics tested. The P. maltophiliu strain (PRS) was resistant to all of the antibiotics tested except chloramphenicol at 10 pg/ml and colistin sulphate at 10 Mg/ml. The strain of Bacillus sp. (PR2) was sensitive to all of the antibiotics except oxolinic acid (10 pg/ml) to which it was resistant. Therapy Five prawns from the Institute of Aquaculture stock (of mean weight 4.8 g), two with heavy and three with moderate burn spot lesions, were exposed for 1 h to 10 &g/ml oxolinic acid in water. Further progress of the burn spot lesions was arrested in the moderately infected animals, so that the next post-treatment moult removed all evidence of lesions. The effectiveness of the treatment on the severely affected animals was less evident

Transport water Telson lesion prawn M3 Walking leg lesion prawn M4 Teloon lesion prawn M5

PR7

-

-

-

-

Gram stain

R

R

R

R

Shape

Primary tests O/F

F F

o+

F

Motility + +

+

+

R, rod shaped; 0, oxidative; F, fermentative.

PRlO

PR9

PR8

Source

Isolate code

+

+

+

Catalase

+

-

+

+

Oxidase

+

+

+

+

Growth at 37°C

3047127

5002000

3047127

3046125

API profile no.

Presumptive identification of bacterial isolates from prawn burn spot lesions (Malaysian adults)

TABLE 2

Aeromonas

Pseudomonas

Aeromonas

Aeromonas

hydrophila

maltophilia

hydrophila

hydrophila

Identification

165 TABLE 3 Sensitivity* of bacterial isolates to anti-microbial agents Anti-microbial agent

Cont. @g/ml)

2 Ampicillin Ampicillin 25 Chloramphenicol 10 Cloxacillin 5 Colistin sulphate 10 Co-trimoxazole 25 ~rythromyein 10 Fum~o~idone 50 Gentamycin 10 Kanamycin 30 Neomycin 10 Nitrofurantoin 200 Novobiocin 5 Oxolinic acid 10 Oxytetracycline 10 Penicillin G 1.5 units Sulphafurazole 500 Tetracycline 10 Tetracycline 50

Bacterial strains PRl

PR2

PR3

PR4

PR5

PR6

PR7

PR8

PR9

PRlO

R R R R S R R R S s s R R R S R R s s

S

R R S R S S R S S S S S R S R R R R S

R R S R S S S S S S S S R S S R S S S

R R S R S S S S S S S S R S R R S S S

R R s R R S s s s s s s R s s R s s s

R S s R S R s s s s s s R s s R s s s

R R S R R S S S S S S S R S S R S S S

R R S R S R R R R R R R R R R R R R R

R R S R R S R S S S S S R S S R S S S

S S

S S S S S

s S S S S R S S S S S

*Sensitive (S) or resistant (R) to test compound and concentration.

and, therefore, both of these animals were treated again with oxolinic acid 7 days after the first treatment. One of the severely affected animals showed no signs of recovery and died 4 days after the second treatment; the second animal moulted 10 days after that treatment and was then apparently normal. Scanning electron microscopy After the success of this preliminary trial, further moderately infected animals were treated with oxohnic acid in the same way so that the lesions could be examined under the sc~~g electron microscope. Before treatment, the lesions were distinguishable at low magnification as depressed, eroded areas with irregularly cracked and displaced fragments of exoskeleton (Figs. 2, 3 and 4). At high magnifications, these areas were seen to be covered by a heavy coating of bacterial cells (Figs. 5 and 6). The majority of these cells were of uniform appearance, being rods 0.31.0 (-2.0) pm in length and 0.2 pm in width. Many were embedded in an amorphous apparently mucoid material on the lesion surface. Occasional 0.2-0.3 pm in diameter coccoid cells were also present. Areas away from the lesions were almost completely free of bacterial cells.

Fig. 4-6. SEM of telson lesion showing bacterial encrustation X 3000; Fig. 6, X 15 000).

(Fig. 4, x 700; Fig. 5,

167

Fig. 7. SEM of lesion 1 h after oxolinic acid treatment (1 h, 10 ppm) showing initial loss of integrity of bacterial cells (X 17 000).

Figs. 8-9. SEM of lesion 12 h after oxolinic acid treatment showing remaining totally plasmolysed bacterial cells (Fig, 8, X 12 000; Fig. 9, X 6000).

Figs. 10-12. SEMs of representative lesions 21 h after oxolinic acid treatment showing surfaces which are almost entirely free of bacteria but with some recolonization by small coccoid cells (Fig. 10, X 200; Fig. 11, X 2000; Fig. 12, X 2200).

169

The effect of oxolinic acid treatment on the lesions was visible under the scanning electron microscope by the end of the first hour after treatment in that all of the bacterial cells had begun to show a crinkling irregularity of their surface. This condition presumably reflected a loss of turgor (Fig. 7). During the following hours, this effect increased rapidly and the bacterial coating over the lesion began to disappear. By 12 h after the oxolinic acid treatment, bacteria could only be found with difficulty and those remaining were completely flattened and plasmolysed (Figs. 8 and 9). From 12 h to beyond 48 h, the lesions remained almost, if not completely, free of any microbial growth (Figs. 10, 11 and 12). No external signs of lesion repair were evident during this period. DISCUSSION

Burn spot disease is a common condition in crustaceans, particularly in intensive culture (Johnson, 1983). The disease has been reported in all of the stages of cultivated Mucrobruchium and is a cause of mortality in the hatchery phase (Aquacop, $977; Martinez et al., 1982). In South East Asia, unsightly burn spot lesions on adult Mucrobruchium reduce their market value by as much as 75%. The cause of burn spot disease in cultured prawns has been attributed variously to handling, poor water quality and hygiene, overcrowding and dietary deficiency. The scanning electron microscope appearance of the lesions in our experimental animals is consistent with the bacterial aetiology for the condition. The lesions were heavily covered with a deep mat of bacterial cells and treatment with an anti-microbial agent allowed rapid recovery. There is no consensus as to the bacterial species responsible and it must be emphasised that this condition -may well be caused by a range of different micro-organisms at different sites and at different times. Burn spot lesions on marine and freshwater crustaceans in a range of climatic conditions may well have different aetiologies. Certainly, A. hydrophilu, isolated from the 1M. rosenbergii lesions described above, is capable of growth at temperatures ranging from temperate to tropical, is oxolinic acid-sensitive and is capable of chitinoclastic activity (Allen et al., 1983). None of the other predominant bacterial isolates were sensitive to oxolinic acid. It has not yet been possible to undertake pathogenicity experiments with the strains isolated from diseased prawns but the pure growth of A. hydrophilu from alcohol-dipped lesions, its considerable sensitivity to oxolinic acid in vitro and the successful treatment of the disease condition with oxolinic acid in vivo imply that this bacterial strain was the cause of the mortalities. The prawns examined in the second part of the study immediately after import also yielded A. hydrophilu which was oxolinic acid-sensitive in vitro. Thus, A. hydrophilu was found to be a constant inhabitant of exoskeletal lesions of M. rosenbergii both from the laboratory conditions in Britain and the Malaysian farm environment.

170

The aetiology of these lesions needs further investigation but the results obtained in this preliminary study indicate strongly that burn spot lesions are readily susceptible to therapy with suitable anti-microbial agents. Once the infecting and encrusting bacteria are destroyed, the ability of M, rosenbergii to recover and repair the damage is seen to be great. Since a major factor in the commercial effects of these infestations in intensive culture is a loss of value because of the unsightliness of the infected animals, the use of anti-microbial agents to control chitinoclastic bacterial infections is of obvious potential. In this preliminary trial, oxolinic acid was selected for scanning electron microscope examination because of its low toxicity to fish (Austin et al., 1983) and because the compound has recently been licensed for fisheries use in the U.K. The fact that effective therapy can be achieved with oxolinic acid by simple short bath technique makes practicable the possibility of employing treatments of this type during routine husbandry movements of stock rather than by the use of long-term baths or by incorporation into food. ACKNOWLEDGEMENT

We are most grateful to Prof. Ang Kok Jee of the Universiti Pertanian, Malaysia, for supplying the prawns used in this study.

REFERENCES Allen, D.A., Austin, B. and Colwell, R.R., 1983. Numerical taxonomy of bacterial isolates associated with a freshwater fishery. J. Gen. Microbial., 129: 2043-2062. Aquacop, 1977. Observations on diseases of crustacean cultures in Polynesia. Proc. World Maricult. Sot., 8: 685-703. Austin, B., Morgan, D.A. and Alderman, D.J., 1981. Comparison of anti-microbial agents for the control of vibriosis in marine fish. Aquaculture, 26: l-12. Austin, B., Rayment, J. and Alderman, D.J., 1983. Control of furunculosis by oxolinic acid. Aquaculture, 31: 101-108. Ayres, P. and Edwards, E., 1982. Notes on the distribution of “black spot” shell disease in crustacean fisheries. Chem. Ecol., 1: 125-130. Brock, J., 1983. Diseases (infectious and non-infectious), metazoan parasites, predators and public health considerations in Macrobrachium culture and fisheries. In: J.P. McVey (Editor), Handbook of Mariculture, I, Crustacean Aquaculture. CRC Press, Boca Raton, Florida, pp. 329-370. Burns, C.D., Berrigan, M.E. and Henderson, G.E., 1979. Fusarium sp. infections in the freshwater prawn Macrobrachium rosenbergii (De Man). Aquaculture, 16: 193-198. Cowan, S.T. and Steel, K.J., 1974. Manual for the Identification of Medical Bacteria. Cambridge University Press, Cambridge, 238 pp. Doughtie, D.G. and Rao, K.R., 1983. Ultrastructural and histological study of degenerative changes leading to black gills in grass shrimp exposed to a dithiocarbamate biocide. J. Invertebr. Pathol., 41: 33-50. Doughtie, D.G., Conklin, P.J. and Rao, K.R., 1983. Cuticular lesions induced in grass shrimp exposed to hexavalent chromium. J. Invertebr. Pathol., 42: 249-258.

171 Gopalan, U.K. and Young, J.S., 1975. Incidence of shell disease in shrimp in the New York Bight. Mar. Pollut. Bull., 6: 149-153. Johnson, P.T., 1983. Viral, bacterial and fungal diseases. In: D.E. Bliss (Editor), The Biology of Crustacea, 6, Pathobiology. Academic Press, New York, pp. l-78. Lightner, D.V., 1984. Diseases of cultured penaeid shrimp. In: J.P. McVey (Editor), Handbook of Mariculture, I, Crustacean Aquaculture. CRC Press, Boca Raton, Florida, pp. 289-320. Lightner, D.V., Colvin, L.B., Brand, C. and Ranald, D.A., 1977. Black death, a disease syndrome of penaeid shrimp related to a dietary deficiency of ascorbic acid. Proc. World. Maricult. Sot., 8: 611-623. Lightner, D.V., Hunter, B., Margarelli, P.C., Jr. and Colvin, L.B., 1979. Ascorbic acid: nutritional requirement and role in wound repair in penaeid shrimp. Proc. World Maricult. Sot., 10: 513-528. Martinez, L.E., Molinares, A.M., Villanueva, J. and Conroy, D.A., 1982. Preliminary observations on the application of nifurpirinol for the control of potential disease problems in Mucrobrachium acanthurus. In: M.B. New (Editor), Giant Prawn Farming. Elsevier Scientific Publishing Company, Amsterdam, pp. 285-289. New, M. and Singholka, S., 1982. Freshwater prawn farming - a manual for the culture of Macrobrachium rosenbergii. FAO Fish. Tech. Pap,, 225: l-116. Roald, S.O., Aursj$, J. and Hastein, T., 1981. Occurrence of shell disease in lobsters (Homarus gammarus L.) in the southern part of Oslofjord, Norway. Fiskeridir. Skr. Ser. Havunders., 17: 153-160: Rosen, B., 1970. Shell disease of aquatic crustaceans. Symposium on Diseases of Fishes and Shellfishes. Am. Fish. Sot. Spec. Publ., 5: l-526. Schlotfeldt, J.H., 1972. Jahreszeitliche Abhangkeit der “Schwarzfleckenkrankheit” bei der Gamele, Crungon crangon (L.). Ber. Dtsch. Wiss. Komm. Meeresforsch., 22: 397-399. Sindermann, C.J., 1977. Disease diagnosis and control in North American aquaculture. Developments in Aquaculture and Fisheries Science, 6. Elsevier Scientific Publishing Company, Amsterdam, 330 pp. Young, J.S. and Pierce, J.B., 1975. Shell disease in crabs and lobsters from New York Bight. Mar. Pollut. Bull., 6: 101-105.